Water-soluble compositions of bioactive lipophilic compounds

ABSTRACT

Water-soluble compositions comprising a lipophilic compound and a solubilizing agent of the general formula: 
     
         {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }.sub.p --Y        (I) 
    
     wherein: 
     X is a residue of a hydrophobic moiety, 
     Y is a residue of a hydrophilic moiety, 
     p is 1 or 2, 
     m is 0 or 1, and 
     n is an integer greater than or equal to 0 
     are disclosed. The lipophilic compound is preferably selected from the group consisting of water-insoluble ubiquinones, ubiquinols, vitamins, provitamins, polyene macrolide antibiotics, and mixtures thereof. The hydrophobic moiety is preferably a sterol or a tocopherol and the hydrophilic moiety is preferably a polyalkylene glycol. In preferred embodiments, the sterol is cholesterol or sitosterol, the tocopherol is a-(+)-tocopherol, the polyalkylene glycol is a polyethylene glycol or its methyl monoether having an average molecular weight between 600 and 1000, p is equal to 1 or 2, m is equal to 0 or 1 and n is an integer between 2 and 18.

FIELD OF THE INVENTION

The present invention relates to water-soluble compositions of bioactivelipophilic compounds, to compounds useful for the preparation of suchcompositions, to methods of preparing such compounds and compositions,and to the use of such compositions as therapeutics and cosmetics.

BACKGROUND OF THE INVENTION

Many bioactive compounds are highly lipophilic (hydrophobic), meaningthat they are soluble in lipids (oils) and some organic solvents, whilebeing substantially insoluble or only sparsely soluble in water. Thelack of solubility of a bioactive compound in aqueous media is animportant factor limiting its therapeutic applications, making difficultan efficient administration of the compound to a patient. Whenadministered in the form of an oil solution or some kind of water and/oroil suspension or emulsion, lipophilic compounds usually show a poorbioavailability, meaning a low concentration and a long build-up time ofthe compound in the systemic circulation. This lack of bioavailabilityis usually independent of the administration route (topical, oral, orparenteral).

Various approaches to overcoming this limitation are known in the priorart. One known approach consists of dissolving a lipophilic compound ina water-miscible organic solvent, such as ethanol or propylene glycol.When such a solution is admixed with blood or gastrointestinal fluids,however, the lipophilic compound usually precipitates as a solid orliquid emulsion, with a resulting low bioavailability. Furthermore, formany lipophilic compounds no organic, water-miscible solvents exist.Another approach consists of incorporating lipophilic compounds intovarious compositions, frequently inhomogeneous, multiphase emulsions,containing oils and solvents in combination with surfactants. Thesecompositions may improve the bioavailability of the compound withoutsignificantly increasing its solubility in aqueous media, but arenormally suitable only for a particular administration form, usually fortopical applications. Such compositions, which may also induce aprotective immune response in mammals, are of little value fortherapeutic uses where administration of the compound by ingestion orinjection is necessary and where an aqueous solution or a water-solublesolid composition is frequently the only acceptable administration form.

Several approaches to preparing homogenous aqueous solutions oflipophilic bioactive compounds are also known in the prior art. Onemethod consists of preparing a derivative or an analog of a lipophiliccompound having a better solubility in water than the original compound.In the simplest case, this derivative may be a water-soluble salt of thecompound, which salt usually retains the original biological activity,but this approach is applicable only to compounds having acidic or basicproperties. If more substantial modifications are introduced into theoriginal compound to improve its solubility, a decrease or even acomplete loss of the original bioactivity of the compound is frequentlyobserved.

Another method of solubilization consists of preparing a water-solublederivative capable of liberating the original bioactive compound underphysiological conditions. Such derivatives, also known as pro-drugs,usually improve bioavailability of the compound and may also ensure atargeted delivery of the compound or its sustained release over a periodof time. However, this approach usually relies on the presence ofcertain functional groups in the original compound, so it is notuniversally applicable. In addition, synthetic methods of improvingsolubility of a compound by chemical modifications are relativelycomplicated and expensive.

Still another approach to solubilization of bioactive lipophiliccompounds relies on formation of water-soluble complexes. An example arecomplexes with amphipathic compounds containing moieties of two opposingsolubility tendencies (lipophilic and hydrophilic). Such compounds areoften soluble both in organic solvents and in water, so that thesolubilization is usually achieved by dissolving a bioactive lipophiliccompound and an amphipathic compound in a suitable water-miscibleorganic solvent and diluting the solution with water. In some cases theorganic solvent is partially or entirely removed from the original orwater-diluted solution by evaporation or lyophilization and theconcentrate reconstituted with a suitable aqueous medium, withoutprecipitation of the water-insoluble lipophilic compound. When theauxiliary organic solvent cannot be completely removed from thecomposition, this solvent must be pharmaceutically acceptable, whichlimits the choice of applicable solvents.

Bioactive lipophilic compounds in need of solubilization belong tovarious therapeutic categories, such as vitamins (e.g., Vitamin E),antibiotics, in particular macrolide polyene antibiotics(amphotericin-B, nystatin, candicidin), free radicals scavengers (e.g.,tocopherols, ubiquinones), immunosuppressants (e.g., cyclosporine), etc.Various approaches to achieve the solubility and improve thebioavailability of these and other lipophilic compounds are known in theprior art, including formation of water-soluble complexes.

U.S. Pat. No. 5,686,110 discloses water-soluble complexes ofwater-insoluble compounds, including amphotericin-B and cyclosporine,with a polyalkylene oxide polymers end-capped with an alkyl or olefinicgroup, which polymers are soluble both in water and in organic solvents.The water-soluble complexes, which are formed only in the presence of anauxiliary organic solvent, are lyophilized and reconstituted with abuffer solution. The reconstituted aqueous solutions show only a limitedstability, depending mostly on the pH of the solution. Furthermore, theuse of methoxypolyethylene glycol polymers of relatively high molecularweight (2,000 to 5,000) as preferred solubilizing agents increases theamount by weight of polymer necessary for solubilization of thebioactive compound.

U.S. Pat. No 5,798,333 discloses a homogenous, water-soluble composition(concentrate) of cyclosporine, which can be diluted with an aqueoussolution without precipitation of cyclosporine. The concentratecomprises cyclosporine and tocophersolan (polyoxyethanyl-a-tocopherylsuccinate, TPGS) dissolved in a hydrophilic organic solvent, such aspropylene glycol. Solvent-free compositions are not disclosed, as theywould likely be unstable or in homogeneous.

WO 96/17626 discloses water-soluble compositions of ubiquinonescomprising polyoxyethanyl-cholesteryl sebacate (PCS) as a solubilizingagent. A ubiquinone, in particular Coenzyme Q₁₀, is solubilized bydissolving both Coenzyme Q₁₀ and PCS in tetrahydrofuran at anapproximate molar ratio of 1:3 and diluting this solution with water.The solution is then evaporated to dryness under reduced pressure andreconstituted with a suitable buffer solution.

Coenzyme Q₁₀ (CoQ10) is a natural compound whose therapeutic potentialhas been recently recognized for a number of disorders, includingcongestive heart failure, muscular distrophy, periodontal disease,correction of drug-induced deficiencies, and immune restoration (AIDS,allergies), to name a few. Coenzyme Q₁₀ is also of great interest tocosmetic industry, since it can be included into cosmetic preparationsas agent slowing down natural skin ageing processes. The biologicalactivity of Coenzyme Q₁₀ is believed to be linked to its ability to actas an antioxidant and free radical scavenger protecting integrity ofcell membranes and to offset the inability of diseased cells tomanufacture sufficient energy for cellular repair, by stimulatingmitochondrial respiration and production of ATP. For effectiveness inboth clinical and cosmetic applications, preparations of Coenzyme Q₁₀with high bioavailability and solubility in aqueous media are usuallyrequired.

Even though various methods of improving solubility of lipophiliccompounds, such as Coenzyme Q₁₀, in aqueous media are known in the priorart, they are not equal in terms of simplicity, scope of applicability,stability of the prepared formulations, etc. The present inventionprovides a new method of solubilization of lipophilic compounds, whichis free of many prior art drawbacks and limitations.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides a water-solublecomposition comprising a lipophilic compound and a solubilizing agent ofthe general formula:

    {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }--Y               (I)

wherein:

X is a residue of a hydrophobic moiety,

Y is a residue of a hydrophilic moiety,

p is 1 or 2,

m is 0 or 1, and

n is an integer greater than or equal to 0.

The lipophilic compound is preferably selected from the group consistingof ubiquinones, ubiquinols, vitamins, provitamins, polyene macrolideantibiotics, and mixtures thereof. The hydrophobic moiety is preferablya sterol or a tocopherol and the hydrophilic moiety is preferably apolyalkylene glycol. In preferred embodiments, the sterol is cholesterolor sitosterol, the tocopherol is a-(+)-tocopherol, the polyalkyleneglycol is a polyethylene glycol or its methyl monoether having anaverage molecular weight between 600 and 1000, p is equal to 1 or 2, mis equal to 0 or 1 and n is an integer between 2 and 18.

According to another aspect, the invention provides a method ofpreparation and purification of a water-soluble composition comprising asolubilizing agent of the general formula (I), in the presence or in theabsence of an auxiliary organic solvent. The solubilization is achievedby dissolving a water-insoluble lipophilic compound and a solubilizingagent in a water-miscible organic solvent, diluting the solution withwater, and removing the organic solvent and optionally a part of waterunder reduced pressure. Alternatively, a lipophilic compound andsolubilizing agent can be admixed directly in a predetermined molarratio and heated to a temperature higher than the their respectivemelting points, to form a water-soluble composition in the form of aclear melt, which can be then diluted with an aqueous solution to adesired concentration. The water-soluble composition may be additionallypurified by dissolving it in a small amount of water, heating thesolution until the composition separates as a clear liquid phase, andremoving the separated composition from the solution while keeping thetemperature of the solution substantially unchanged.

According to still another aspect, the invention provides pharmaceuticalor cosmetic formulations comprising a water-soluble composition of alipophilic compound and a solubilizing agent of formula (I).

According to yet another aspect, the invention provides novelsolubilizing agents of formula (I) and methods of their preparation andpurification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the toxicity of various solubilizing agents.

FIG. 2 is a graph showing the bioavailability of CoQ₁₀ in variouswater-soluble compositions.

FIG. 3 is a graph showing the effect of PTS-CoQ₁₀ composition oncellular ATP level in NT2 cells.

FIG. 4 is a graph showing the protective effect of PTS-CoQ₁₀ compositionagainst hypoxia in NT2 cells.

FIG. 5 is a graph showing the effect of PTS-CoQ₁₀ and PTS-Vitamin Ecompositions on the viability of hypoxia-treated NT2 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides a method of solubilization of substantiallywater-insoluble, bioactive lipophilic compounds, by providing awater-soluble composition comprising the lipophilic compound and asolubilizing agent of the following general formula:

    {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }.sub.p --Y        (I)

wherein:

X is a residue of a hydrophobic moiety,

Y is a residue of a hydrophilic moiety,

p is 1 or 2,

m is 0 or 1, and

n is an integer greater than or equal to 0.

The hydrophobic moiety of the solubilizing agent is a hydrophobic(lipophilic) molecule having an esterifiable hydroxy group and ispreferably a sterol or a tocopherol, in particular cholesterol,7-dehydrocholesterol, campesterol, sitosterol, ergosterol, stigmasterol,or an a-, b-, g-, or d-tocopherol. Cholesterol and sitosterol arepreferred sterols, sitosterol being particularly preferred.a-(+)-Tocopherol and a-(±)-tocopherol are preferred tocopherols,a-(+)-tocopherol (vitamin E) being particularly preferred. The residueof the hydrophobic moiety is the entire hydrophobic molecule, except forits esterified hydroxy group, such as 3-b-hydroxy group of cholesterolor sitosterol or 6-hydroxy group of a-tocopherol.

The hydrophilic moiety of the solubilizing agent is a hydrophilicmolecule having an esterifiable hydroxy or carboxy group, and ispreferably selected from the group consisting of polyalcohols,polyethers, polyanions, polycations, polyphosphoric acids, polyamines,polysaccharides, polyhydroxy compounds, polylysines, and derivativesthereof. Of those, polyethers are preferred, polyalkylene glycols beingparticularly preferred. The term "polyalkylene glycol" includes polymersof lower alkylene oxides, in particular polymers of ethylene oxide(polyethylene glycols) and propylene oxide (polypropylene glycols),having an esterifiable hydroxy group at least at one end of the polymermolecule, as well as derivatives of such polymers having esterifiablecarboxy groups. The residue of the hydrophilic moiety is the entirehydrophilic molecule, except for its esterified hydroxy or carboxy groupor groups, such as terminal hydroxy groups of a polyethylene glycolmolecule.

Polyethylene glycols are most particularly preferred for the practice ofthe present invention. Suitable polyethylene glycols may have a freehydroxy group at each end of the polymer molecule, or may have onehydroxy group etherified with a lower alkyl, e.g., a methyl group. Alsosuitable for the practice of the invention are derivatives ofpolyethylene glycols having esterifiable carboxy groups. Polyethyleneglycols are commercially available under the trade name PEG, usually asmixtures of polymers characterized by an average molecular weight.Polyethylene glycols having an average molecular weight from about 300to about 5000 are preferred, those having an average molecular weightfrom about 600 to about 1000 being particularly preferred.

Compounds of formula (I) for which m is equal to 1 can be regarded asdiesters of an alkanedioic acid of the general formula HOOC--(CH₂)_(n)--COOH. For the practice of the present invention, alkanedioic acidswith n from 0 to 18 are preferred, those with n from 6 to 10 beingparticularly preferred. Sebacic acid (n=8) is most particularlypreferred.

Bioactive lipophilic compounds which can be solubilized usingsolubilizing agents of the present invention belong to a variety oftherapeutic and chemical categories and include ubiquinones, ubiquinols,sterols, vitamins, provitamins and macrolide polyene antibiotics.

Preferred ubiquiniones and ubiquinols are those of the formula: ##STR1##wherein R is ##STR2## and k is an integer of from 6 to 12. Those with kequal to 10 (ubiquinone-50 or Coenzyme Q₁₀ and ubiquinol-50 or reducedCoenzyme Q₁₀, respectively) are particularly preferred.

Cholesterol, sitosterol, ergosterol and 7-dehydrocholesterol arepreferred sterols. Vitamins A, D, E, and K are preferred vitamins andprovitamin A (b-carotene) is a preferred provitamin. Amongst macrolidepolyene antibiotics, amphotericin-B, nystatin, and candicidin arepreferred.

The water-soluble compositions of the present invention contain abioactive lipophilic compound and a solubilizing agent in a molar ratioof approximately 1:1 to 1:5. The molar ratio of about 1:2 is preferred.The upper limit of the molar ratio is not critical, and the solubilizingagent can be used in any excess. This is not desirable, however, asincreasing the amount of the solubilizing agent decreases theconcentration of the active ingredient in the composition and in itsaqueous solutions.

The water-soluble compositions of the present invention can be preparedby two different procedures, either in the presence or in the absence ofan auxiliary organic solvent. In the first case, a lipophilic compoundand a solubilizing agent are first dissolved in a predetermined molarratio in a water-miscible organic solvent and this solution is thendiluted with a predetermined amount of water, without precipitation ofthe lipophilic compound. The organic solvent and water are then removedby evaporation under reduced pressure. A volatile organic solvent isusually removed first, followed by water, in which case the amount ofwater removed from the solution may be controlled, to achieve a desiredconcentration of the water-soluble composition in the remainingconcentrate. Alternatively, both the organic solvent and water areremoved by evaporation, and the waxy residue is reconstituted with asuitable aqueous medium (such as water, physiological saline, or abuffer solution), to provide a clear aqueous solution.

The organic solvent used is in the above procedure should be a goodsolvent for both the lipophilic compound and the solubilizing agent andhas to be miscible with water. If a water-soluble compositions is to beused in a pharmaceutical formulation, this solvent should be alsopharmaceutically acceptable, as the removal of the solvent byevaporation may not always be possible. Examples of solvents suitablefor the practice of the invention are tetrahydrofuran, ethanol, ethyleneglycol, propylene glycol, and acetic acid. Solvents with a low boilingpoint, such as tetrahydrofuran, are preferred.

The amount of the organic solvent is not critical, and is equal to orgreater than the minimum amount of solvent necessary to dissolve thegiven amounts of the lipophilic compound and solubilizing agent. Theamount of water used for the dilution is also not critical, and ispreferably between 10 to 25 times the volume of the organic solvent.

An alternative procedure for preparing water-soluble compositionsaccording to the invention consists of preparing first a mixture of alipophilic compound and a solubilizing agent in a predetermined molarratio. This mixture is then heated to a temperature higher than therespective melting points of the lipophilic compound and thesolubilizing agent, for a time necessary to obtain a clear melt, whichprocess can be seen as a dissolution of the lipophilic compound in thesolubilizing agent. The melt so obtained can be reconstituted with apredetermined amount of a suitable aqueous medium, to provide a clearaqueous solution of a desired concentration. This method of preparingwater-soluble compositions of the invention is preferred, as it issimpler and avoids limitations of the procedure that relies on anauxiliary organic solvent, such as the pharmaceutically acceptablecharacter of the solvent required for most applications.

The ability of solubilizing agents of the present invention to dissolvelipophilic compounds in the absence of an auxiliary organic solvent canbe used for preparing water-soluble forms of bioactive compounds, inparticular Coenzyme Q₁₀, without purifying it by crystallization aftersynthesis, thus reducing the overall cost of preparing water-solublecompositions of this compound.

Many compositions of the present invention show a decreasing solubilityin water with increasing temperature of the solution. This provides analternative method of isolation and/or purification of suchcompositions. For the purpose of purification, the composition isdissolved in water at a ratio of the composition to water not exceeding1:2 by volume and the solution is heated, for example in a boiling waterbath, for a time necessary to achieve the separation of thewater-soluble composition as a liquid phase, usually a few minutes. Theoily phase is then separated from the hot solution, while keeping thetemperature of the solution substantially unchanged, as cooling of thesolution would increase the solubility of the composition and result ina reduced yield of the recovery. A speedy separation of the oily phaseto avoid the cooling can be achieved, for example, by centrifugation.

The water-soluble compositions of the present invention have a waxyconsistence and may be difficult to manipulate in this highlyconcentrated form. To make them more easily processable as solids, theymay be combined with a suitable solid, water-soluble additives, such asvitamin C, gelatin, a protein supplement, or a polyethylene glycol. Inthe latter case, a polyethylene glycol having an average molecularweight greater than about 5000 is preferred. The ratio of thecomposition and the additive is not critical, but will be usuallylimited to avoid an unnecessary dilution of the active ingredient by theadditive. Such solid composition are particularly useful for thepreparation of certain dosage forms of bioactive compounds, such astablets or granulates.

The compositions of the present invention show an excellent solubilityin water and allow the preparation of aqueous solutions of almost anyconcentration. As the concentrated solutions can be diluted with anaqueous medium in any proportion and over a wide range of pH conditionswithout precipitation of the lipophilic compound, the solubility of thecompound is maintained under physiological conditions, for example afteran oral or parenteral administration of the composition. This normallyresults in an improved bioavailability of the compound.

The compositions of the present invention and aqueous solutions thereofshow an excellent stability over long periods of time (several months atroom temperature, at least one year when refrigerated, or indefinitelywhen frozen) and over wide ranges of temperature and pH conditions(temperatures from -80° C. to 120° C., pH from 2.0 to 8.0). Aqueoussolutions can be repeatedly frozen and thawed without any perceptibledegradation. The stability under high temperature conditions allows aneasy sterilization of the solutions, without compromising the solubilityof the active ingredient.

The water-soluble compositions of the present invention can be easilyincorporated into pharmaceutical or cosmetic formulations, which arethen charcterized by an improved bioavailability of the lipophilicactive ingredient. Such formulations may further contain additionalactive ingredients and/or a pharmaceutically or cosmetically acceptableadditives or vehicles, including solvents, adjuvants, excipients,sweeteners, fillers, colorants, flavoring agents, lubricants, binders,moisturizing agents, preservatives and mixtures thereof. Theformulations may have a form suitable for a topical (e.g., a cream,lotion, gel, ointment, dermal adhesive patch), oral (e.g., a capsule,tablet, caplet, granulate), or parenteral (e.g., suppository, sterilesolution) administration. Among the acceptable vehicles and solventsthat may be employed for administration by injection are water, mildlyacidified water, Ringer's solution and isotonic sodium chloridesolution.

Among water-soluble compositions and pharmaceutical and cosmeticformulations, those comprising Coenzyme Q₁₀ are of particular interest.Coenzyme Q₁₀ is a natural compound whose therapeutic potential has beenrecently recognized for a number of conditions and disorders related tomitochondrial dysfunctions and/or tissue damage caused by free radicalsand oxidants. These include, but are not limited to: cardiovasculardiseases (e.g., congestive heart failure), muscular disorders (e.g.,muscular dystrophy), mitochondrial encephalomyolopaties (i.g., MELAS,KSS), neurodegenerative disorders (e.g., Alzheimer', Parkinson's,Huntington's diseases), restoration of immune deficiencies caused bydrugs or infections (AIDS, allergies, cancer). Of particular interest isthe clinical use of Coenzyme Q₁₀ to minimize tissue damage resulted fromischemia/reperfusion (e.g., stroke, head trauma, angioplasty, organtransplantation, surgery in general). Another area of interest is theuse of Coenzyme Q₁₀ in therapy as adjuvant, for example for infectiousdiseases, in combination with cholesterol-lowering drugs for thetreatment of hypercholesteremia, or in combination with chemotherapeuticagents in the treatment of cancers. Coenzyme Q₁₀ is also of greatinterest to cosmetic industry, as an agent slowing down natural skinageing processes. Water-soluble compositions comprising a macrolidepolyene antibiotic, such as amphotericin-B, nystatin, or candicidin, areof particular interest for the treatment of fungal infections, includingfungal infections in immunocompromised patients.

Bioactive lipophilic compounds in the form of a water-solublecomposition according to the present invention may be administered to awarm-blooded animal, particularly a human, in need of the prophylaxis ortherapy. The dose of a bioactive lipophilic compound and thecorresponding dose of its water-soluble composition for treating theabove-mentioned diseases or disorders vary upon the manner ofadministration, the age, sex, the body weight of the subject, and thecondition being treated, and will be ultimately decided by the attendingphysician or veterinarian. Such an amount of the bioactive compound inthe form of its water-soluble composition as determined by the attendingphysician or veterinarian is referred to herein as a "therapeuticallyeffective amount".

The biological activity of Coenzyme Q₁₀ is believed to be linked to itsability to act as an antioxidant and free radical scavenger protectingintegrity of cell membranes and to offset the inability of diseasedcells to manufacture sufficient energy for cellular repair, bystimulating the mitochondrial respiration and production of ATP. Foreffectiveness in both clinical and cosmetic applications, preparationsof Coenzyme Q₁₀ with high bioavailability and solubility in aqueousmedia are normally desirable.

Most pharmaceutical and cosmetic formulations proposed to date forCoenzyme Q₁₀ suffer from a number of drawbacks, such as the use ofcomponents causing an undesirable immune response when administeredparenterally or gastric disorders when administered orally. Someformulations are stable only over a narrow range of pH values and mostare characterized by a poor bioavailability of the active component(Coenzyme Q₁₀). In particular, there are no reliable formulations forintravenous administration of Coenzyme Q₁₀, as there are no formulationsuniversally applicable to topical, oral, and parenteral administrationroutes. The water-soluble compositions of the present inventioncomprising Coenzyme Q₁₀ are free of the above drawbacks and can beincorporated into pharmaceutical formulations suitable for alladministration routes, topical, oral, and parenteral, which are stableand characterized by a good solubility and bioavailability of the activecomponent.

The invention further provides novel solubilizing agents of the generalformula

    {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }.sub.p --Y        (I)

wherein:

X is a residue of a hydrophobic moiety,

Y is a residue of a hydrophilic moiety,

p is 1 or 2,

m is 0 or 1, and

n is an integer greater than or equal to 0,

provided that:

when p and m are both equal to 1 and the hydrophobic moiety ischolesterol, n is greater than 4 and not equal to 8, and

when p and m are both equal to 1 and the hydrophobic moiety isa-(+)-tocopherol, n is not equal 2,

and wherein X and Y have the earlier discussed meaning.

Compounds excluded by the proviso are known in the prior art, inparticular polyoxyethanyl-cholesteryl sebacate (PCS) andpolyoxyethanyl-a-tocopheryl succinate (TPGS).

The compounds of formula (I) can be prepared by standard methods ofsynthetic organic chemistry, well known to those skilled in the art. Inparticular, compounds where p is equal to 1 or 2 and m is equal to 1 canbe prepared by reacting a compound of the general formula X--OH with acompound of the general formula Z--OC--(CH₂)_(n) --CO--Z, where Z is aleaving group, and further reacting the product so obtained with acompound of the general formula HO--Y--OR, wherein R is hydrogen or analkyl, and X, Y and n are as defined hereinbefore. Halogens, inparticular Cl and Br, are preferred as the leaving group Z. Hydrogen anda lower alkyl (C₁ -C₄) are preferred for R.

Many solubilizing agents of formula (I) show a decreasing solubility inwater with the increasing temperature of the solution, which provides aconvenient method of purification of these compounds. The steps andconditions of the purification process are substantially the same asthose discussed above for the purification of water-soluble compositionsof the invention.

Various aspects of the present invention will be further illustrated bythe following non-limiting examples.

EXAMPLES

The following abbreviations are used throughout the Examples:

CoQ₁₀, CoQ₁₀ --Coenzyme Q₁₀

PCS--polyoxyethanyl-cholesteryl sebacate

PTS--polyoxyethanyl-a-tocopheryl sebacate

PSS--polyoxyethanyl-sitosteryl sebacate

PTD--polyoxyethanyl-a-tocopheryl dodecanodioate

PTSr--polyoxyethanyl-a-tocopheryl suberate

PTAz--polyoxyethanyl-a-tocopheryl azelaate

TPGS--polyoxyethanyl-a-tocopheryl succinate

A number following one of the above abbreviations (e.g., PCS-600)indicates an average molecular weight of the polyoxyethanyl moiety ofthe compound. A number followed by Me abbreviation (e.g., PTS-750Me)indicates a polyoxyethanyl moiety capped with a methyl group(methoxypolyoxyethanyl).

Examples 1 and 2 illustrate methods of preparation of solubilizingagents of the present invention.

Example 1 Preparation of Polyoxyethanyl-sitosteryl Sebacate (PSS-600)

0.83 g of b-sitosterol (Sigma Chem. Co., product #S-5753, approximately60%) was dissolved in 3 ml of dry toluene at 40° C., followed byaddition of 1.33 mmole of triethylamine (TEA). 1.33 mmole of sebacoylchloride dissolved in 2 ml of dry toluene was than added (dropwise,while stirring, and under anhydrous conditions) to the b-sitosterol-TEAsolution. The reaction was carried out for 10 min at room temperature,at which time 2 mmole of PEG-600 (polyethylene glycol, Sigma Chem. Co.,product # P-3390) and 2.66 mmole of TEA dissolved in 3 ml of dry toluenewere added dropwise to the reaction mixture. The reaction was continuedwith stirring for additional 20 min at room temperature and the reactionmixture was extracted four times with 3 ml each time of saturatedsolution of NaCl. The toluene was removed under reduced pressure leavinga waxy residue. This product was dissolved in 15 ml of water andwater-insoluble materials removed by filtration. The filtrate waslyophilized, yielding 0.8 g of pale-yellow waxy product (PSS-600). Thesame method was used for the preparation of polyoxyethanyl-cholesterylsebacate (PCS-600).

Example 2 Preparation of Polyoxyethanyl-a-tocopheryl Sebacate (PTS-600)

A solution of 1 mmole of a-tocopherol (Sigma Chem. Co., product #T-3251) and 1.33 mmole of TEA in 3 ml of dry toluene was added(dropwise, under anhydrous conditions, while stirring) to 1.33 mmole ofsebacoyl chloride dissolved in 2 ml of dry toluene. The reaction wascarried out for 10 min at room temperature, followed by a dropwiseaddition of 2 mmole of PEG-600 (polyethylene glycol, Sigma, P-3390) and2.66 mmole of TEA dissolved in 3 ml of toluene. The reaction wascontinued for additional 20 min at room temperature with constantstirring. The reaction mixture was extracted four times with 3 ml eachtime of saturated solution of NaCl and toluene evaporated under areduced pressure. The product was dissolved in 5 ml of water and theresidual toluene was further removed by co-evaporation with water undera reduced pressure. The final waxy product (1.15 g) was obtained bylyophilization.

Other solubilizing agents (Table 1) were obtained by linkingpolyethylene glycol (average molecular weight 1000, Sigma Chem. Co.,product # P-3515) or methoxypolyethylene glycol (average molecularweight 750, Sigma Chem. Co., product # M-7018) to a-tocopherol usingadipoyl, suberoyl, azelaoyl or dodecanedioyl dichlorides. They weresynthesized according to the method of Example 2 and their identitieswere confirmed by mass spectrometry analysis applying MALDI-TOFtechnique.

Example 3 provides molecular characteristics of the synthesizedsolubilizing agents.

Example 3 Molecular Characteristic of Solubilizing Agents Obtained byMALDI-TOF Mass Spectrometry

                  TABLE 1                                                         ______________________________________                                        Molecular mass of synthesized solubilizing agents                                            Molecular mass                                                 Solubilizing agent                                                                           m/z                                                            ______________________________________                                        PSS-600        1194.3 ± 44                                                 PCS-600        1166.1 ± 44                                                 PTS-600        1209.7 ± 44                                                 PTD-600        1237.5 ± 44                                                 PTS-750Me      1355.7 ± 44                                                 PTD-750Me      1383.7 ± 44                                                 PTS-1000       1605.9 ± 44                                                 PTSr-600       1181.6 ± 44                                                 PTSr-1000      1578.2 ± 44                                                 PTAz-600       1195.8 ± 44                                                 PTA-600        1153.5 ± 44                                                 PTSc-600       1125.7 ± 44                                                 ______________________________________                                    

Example 4 illustrates a method of purification of solubilizing agents.

Example 4 Purification of Solubilizing Agents

A solubilizing agent prepared according to Examples 1 or 2 was dissolvedin water at 2:1 v/v ratio. The solution was heated in a boiling waterbath for approximately 2 min, until a visible precipitation occurred.This was followed by a brief centrifugation (at least 2000×g) of the hotmixture to achieve separation of the precipitated product which isinsoluble in hot water. The water phase (supernatant) was removed bydecantation leaving a clear pellet of the product containingapproximately 10% of water.

Examples 5 and 6 illustrate a preparation of water-soluble Coenzyme Q10compositions by a direct admixing of the two components.

Example 5 Direct Preparation of Tocopherol-based Water-solubleCompositions of Coenzyme Q₁₀

                  TABLE 2                                                         ______________________________________                                        Components of tocopherol-based water-soluble Coenzyme Q.sub.10                compositions                                                                  ______________________________________                                        Coenzyme Q.sub.10                                                                            0.01              g                                            TPGS           0.035             g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTS-600        0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTD-600        0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTS-750Me      0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTD-750Me      0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTS-1000       0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTSr-1000      0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTSr-600       0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PTAz-600       0.03              g                                            ______________________________________                                    

Table 2 shows amounts of starting materials used for the preparation ofa-tocopherol-based water-soluble compositions of Coenzyme Q₁₀. In eachcase the two components were admixed together at a predetermined ratioand were heated to a temperature higher than their melting points,typically 60° C.-80° C., until the components melted together and formeda clear, transparent and uniform melt. The optimal ratio of asolubilizing agent to Coenzyme Q₁₀ was found to be 2:1 mol/mol or 3:1w/w. These compositions could be stored in sealed vials for at least 2-3years when refrigerated. They could be reconstituted at any time withwater or physiological solution of saline (0.9%), at any ratio, and theyremained water soluble and stable for several month when refrigerated orfrozen.

Example 6 Direct Preparation of Sterol-based Water-soluble Compositionsof Coenzyme Q₁₀

                  TABLE 3                                                         ______________________________________                                        Components of sterol-based water-soluble Coenzyme Q.sub.10                    compositions                                                                  ______________________________________                                        Coenzyme Q.sub.10                                                                            0.01              g                                            PCS-600        0.03              g                                            Coenzyme Q.sub.10                                                                            0.01              g                                            PSS-600        0.03              g                                            ______________________________________                                    

Table 3 shows amounts of starting materials used for the preparation ofsterol-based, water-soluble compositions of Coenzyme Q₁₀. In each casethe two components were admixed at a predetermined ratio (the optimalratio of a solubilizing agent to CoQ10 was found to be 2:1 mol/mol or3:1 w/w) and were heated to a temperature higher than their meltingpoints, typically 60° C.-80° C., until the components melted togetherand formed a clear transparent liquid. The liquid was dissolved in water(at a ratio 2:1 v/v) and the solution was heated in a boiling water bathfor approximately 2 minutes, until clear waxy precipitate was formed.The precipitate could than be separated from the hot solution bycentrifugation at 2000×g. To achieve its full water solubility at roomtemperature, the cycle of heating, centrifugation and cooling to roomtemperature, without removal of supernatant between the cycles, wasrepeated 2 to 3 times. After the final centrifugation, the hotsupernatant was decanted and clear transparent pellet of the product wasrecovered. The compositions could be stored refrigerated in sealed vialsfor at least 2-3 years. Aqueous solutions (in water or saline) of theabove compositions could be prepared and they were stable for severalmonths when refrigerated.

Examples 7, 8 and 9 illustrate preparation of water-soluble Coenzyme Q10compositions in the presence of an auxiliary solvent.

Example 7 Solvent-based Preparation of Water Soluble Compositions ofCoenzyme Q₁₀

                  TABLE 4                                                         ______________________________________                                        Components of water-soluble compositions of Coenzyme Q.sub.10                 ______________________________________                                        Coenzyme Q.sub.10                                                                            5                 g                                            PCS-600        15                g                                            THF            30                ml                                           H.sub.2 O      400               ml                                           Coenzyme Q.sub.10                                                                            0.01              g                                            PSS-600        0.03              g                                            THF            0.1               ml                                           H.sub.2 O      2.5               ml                                           Coenzyme Q.sub.10                                                                            0.3               g                                            PTS-600        1.0               g                                            THF            4.0               ml                                           H.sub.2 O      60.0              ml                                           Coenzyme Q.sub.10                                                                            0.01              g                                            PTD-600        0.03              g                                            THF            0.1               ml                                           H.sub.2 O      2.5               ml                                           Coenzyme Q.sub.10                                                                            0.01              g                                            PTS-750Me      0.03              g                                            THF            0.1               ml                                           H.sub.2 O      2.5               ml                                           Coenzyme Q.sub.10                                                                            0.01              g                                            PTD-750Me      0.03              g                                            THF            0.1               ml                                           H.sub.2 O      2.5               ml                                           ______________________________________                                    

Table 4 shows amounts of starting materials used for variouswater-soluble Coenzyme Q₁₀ compositions. In each case Coenzyme Q₁₀ and asolubilizing agent were both dissolved in tetrahydrofuran (THF) and thesolution was added to water with vigorous stirring, while maintainingthe temperature of the mixture close to 0° C. The solvent and a part ofwater were then evaporated under a reduced pressure to obtain a desiredconcentration of Coenzyme Q₁₀, usually 80-100 mg/ml. These compositionscould be stored refrigerated for at least 2-3 years and could bereconstituted with aqueous media (water, saline) to a desired finalconcentration of Coenzyme Q₁₀ in the water-soluble form. Compositionswith higher concentrations of Coenzyme Q₁₀ (up to 200 mg/ml) were alsoobtained by following this procedure.

Example 8 Solvent-based Preparation of Water-soluble TPGS--Coenzyme Q₁₀Composition

                  TABLE 5                                                         ______________________________________                                        Components of water-soluble TPGS-Coenzyme Q.sub.10                            ______________________________________                                        Coenzyme Q.sub.10                                                                            0.01              g                                            TPGS           0.035             g                                            THF            0.1               ml                                           H.sub.2 O      2.5               ml                                           ______________________________________                                    

Table 5 shows amounts of starting materials used for water-solubleTPGS-Coenzyme Q₁₀ composition which was prepared according to theprocedure described in Example 7. However, this product did notprecipitated from an aqueous solution after heating. Water and thesolvent could be removed by evaporation under a reduced pressure. Watersolubility and stability of the composition was the same as thosedescribed in Examples 4 and 5.

Example 9 describes a procedure for the preparation of water-solubleubiquinol compositions.

Example 9 Preparation of Water-soluble Ubiquinol Compositions

                  TABLE 6                                                         ______________________________________                                        Components of water-soluble ubiquinol compositions                            ______________________________________                                        Ubiquinol 50   0.3               g                                            TPGS           0.9               g                                            Ubiquinol 50   0.3               g                                            TPGS           0.9               g                                            Propylene      0.9               ml                                           Glycol usp.                                                                   Ubiquinol 50   0.1               g                                            Coenzyme Q.sub.10                                                                            0.1               g                                            PTS-600        0.5               g                                            Ubiquinol 50   0.1               g                                            Coenzyme Q.sub.10                                                                            0.1               g                                            Vitamin E      0.001             g                                            PTS-600        0.5               g                                            ______________________________________                                    

Table 6 shows amounts of starting materials used for water-solubleubiquinol (a reduced form of Coenzyme Q₁₀) compositions. Ubiquinol wasfirst prepared by the following method. 0.3 g of Coenzyme Q₁₀ and 0.2 gof zinc dust were suspended in 2.5 ml of glacial acetic acid. Themixture was placed in 50° C. water bath, with occasional shaking, forapproximately 15 min. The reaction mixture was diluted with 2.5 ml ofwater and extracted twice with 5 ml of hexane. The hexane extracts werecombined, washed twice with 2.5 ml of water, dried over anhydrousmagnesium sulfate, evaporated under reduced pressure and finally under ahigh vacuum. The resulting white waxy residue of ubiquinol was combinedwith 0.9 g of TPGS and it was heated to 60° C.-80° C. until the mixturemelted and became clear. Upon reconstitution with water an opaquesolution was formed. This composition remained unchanged for up to 2months when sealed under argon and frozen. The water solubility of thiscomposition could be improved by admixing with propylene glycol at aratio given in the Table 6. The aqueous (water and saline) solutions ofthis composition were stable and could be stored sealed under argon whenfrozen.

Examples 10 and 11 illustrate preparation of water soluble vitamin E andb-carotene compositions.

Example 10 Preparation of Water-soluble Formulations of Vitamins

                  TABLE 7                                                         ______________________________________                                        Components of water-soluble compositions of vitamin E and                     b-carotene                                                                    ______________________________________                                        Vitamin E      0.10              g                                            PTS-600        0.60              g                                            Vitamin E      0.22              g                                            PCS-600        1.00              g                                            THF            2.50              ml                                           H.sub.2 O      35.00             ml                                           Vitamin E      0.025             g                                            PTS-600        0.150             g                                            THF            0.125             ml                                           H.sub.2 O      2.0               ml                                           Provitamin A   0.01              g                                            (b-carotene)                                                                  PTS-600        0.50              g                                            THF            0.20              ml                                           H.sub.2 O      3.0               ml                                           ______________________________________                                    

Table 7 shows amounts of starting materials used for variouscompositions prepared by direct admixing method (Example 5) or with theaid of an auxiliary solvent (Example 7). The optimal ratio of asolubilizing agent to Vitamin E was found to be 2:1 mol/mol or 6:1 w/w.The concentrated compositions could be stored refrigerated for up to 2years. The aqueous solutions (in water or saline) were also stable andcould be stored frozen.

Example 11 illustrates methods for isolation, concentration andsterilization of the compositions according to the present invention.

Example 11 Isolation, Concentration and Sterilization of Water-solubleCompositions

According to the present invention, two processes can be applied for thepreparation of water-soluble compositions of lipophilic compounds: (i) alipophilic compound and a solubilizing agent can either be combineddirectly and melted together, or (ii) the two are first dissolved in awater-miscible auxiliary solvent, the solution mixed with water and boththe solvent and the excess of water are removed by evaporation. Theobtained compositions are soluble in aqueous media at room temperature,but not at temperatures higher than approximately 80° C. Theyprecipitate out of water when heated, but return to the solution uponcooling. This provides a simple, effective and inexpensive method oftheir isolation, concentration and sterilization.

Typically, the aqueous solutions of the compositions (with the exceptionof TPGS-Coenzyme Q₁₀, Example 9) are heated in a boiling water bath forabout 2 min or until visible precipitate is formed. The separation ofthe precipitates from the hot water is accelerated by a rapidcentrifugation at 2000×g g. The water phase is decanted and the obtainedwaxy, transparent composition can be stored refrigerated in sealedvials. It remains stable for at least 8 month and can be further dilutedwith aqueous media (water, saline or a buffer). When refrigerated, thesesolutions are stable for at least 3 years. The process ofprecipitation/solubilization is reversible and repeating it severaltimes improves the stability of diluted aqueous solutions of thecomposition. If precipitation of the composition occurs during storage,its solubility can be restored by applying the aboveheating/centrifugation cycle. The compositions are also suitable forinjections, since the described process can be applied for theirsterilization.

Example 12 illustrates the preparation of dry powder compositionscontaining Coenzyme Q₁₀.

Example 12 Preparation of Dry Powder Coenzyme Q₁₀ Compositions

                  TABLE 8                                                         ______________________________________                                        Components of dry powder Coenzyme Q.sub.10 compositions                       ______________________________________                                        CoQ10/PCS-600 composition                                                                        0.07 g CoQ10/0.21 g PCS-600                                containing 40 mg of CoQ10/ml                                                  of water                                                                      BSA (0.25 g/ml of water)                                                                         0.25 g                                                     CoQ10/PCS-600 composition                                                                        0.07 g CoQ10/0.21 g PCS-600                                containing 40 mg of CoQ10/ml of water                                         Gelatin (0.125 g/ml in water)                                                                    0.25 g                                                     CoQ10/PCS-600 composition                                                                        0.03 g CoQ10/0.09 g PCS-600                                containing 40 mg of CoQ10/ml of water                                         Vitamin C (0.25 g/ml in water)                                                                   0.50 g                                                     ______________________________________                                    

Table 8 shows amounts of starting materials used for the preparation ofdry powder compositions of Coenzyme Q₁₀. The aqueous solutions of theabove components were combined and lyophilized. The resulting powderedproducts could be pressed into tablets which remained stable andwater-soluble.

Example 13 illustrates the preparation of dry powder compositionscontaining multiple components.

Example 13 Preparation of Compositions Containing Coenzyme Q₁₀, VitaminE and Vitamin C

                  TABLE 9                                                         ______________________________________                                        Components of water soluble complex compositions                              ______________________________________                                        Coenzyme Q10/PTS-600 composition                                                                 0.03 g CoQ10/0.09 g PTS-600                                (containing 40 mg of CoQ10/ml                                                 of water)                                                                     Vitamin E/PTS-600 composition                                                                    0.005 g Vit E/0.025 g PTS-600                              (containing 20 mg of vitamin E/ml)                                            Vitamin C (0.25 g/ml in water)                                                                   0.75 g                                                     ______________________________________                                    

Table 9 shows amounts of starting materials used. The aqueous solutionsof the above components were combined and lyophilized. The resultingpowdered products could be pressed into tablets which remained stableand water-soluble.

Example 14 demonstrates the lack of toxicity of solubilizing agents.

Example 14 In Vitro Toxicity Study

Stock solutions of solubilizing agents (1 mg/ml in PBS) were preparedand were added directly to lymphocytic Jurkat and neuroblastoma NT2 cellcultures in the amounts required to achieve a final concentration in themedium of 200, 100 and 50 mg/ml and cell viability was assessed bytrypan blue exclusion assay. The results of assessment are shown in FIG.1.

With the exception of commercially available TPGS, none of thesolubilizing agents of the invention had adverse effects on either cellgrowth or viability in vitro when added to cell cultures atconcentrations up to 200 mg/ml. Also, no toxicity was observed when thecompositions containing CoQ10 and PTS, PCS or PSS were given to SpragueDawley rats at concentrations up to 20 mg/kg body weight, as shown inFIG. 2

Although no rationale can be provided to explain the toxicity of TPGSobserved in the experimental paradigm used, its usefulness as asolubilizing agent in pharmaceutical formulations should be furtherinvestigated and should be taken with caution.

Example 15 illustrates improved bioavailability of water-soluble formsof CoQ10.

Example 15 Bioavailability of Water-soluble Forms of Coenzyme Q₁₀

CoQ10, in the form of either water-soluble or oil-soluble compositions,was administered by gavage to 300-350 g male Sprague Dawley rats at adose of 6 mg/kg body weight. Blood samples were collected and CoQ10content was measured in plasma by the HPLC method which we havedeveloped for analysis of CoQ10 in biological samples (Graves et al.,Analysis of CoEnzyme Q10 content in human plasma and other biologicalsamples. In: Free Radicals and Antioxidant Protocols, Ed. D. Armstrong,Humana Press. pp: 353-365, 1998).

Briefly, 0.1 ml sample of plasma or lysed cells (Examples 15 and 16),was mixed with 0.33 ml of 1-propanol, vortexed for 30 sec and left tostand at room temperature for 1 min. 0.86 ml of n-hexane were thanadded, the sample was vigorously vortexed for 30 sec, and centrifuged toachieve a phase separation and to pellet the denatured proteins. Theupper phase which consisted of n-hexane and 1-propanol and containedCoQ₁₀ was collected and evaporated to dryness under argon. The remainingdry residue was redissolved in 60 ml of ethanol and 2 ml of H₂ O₂ inorder to convert all CoQ₁₀ to its oxidized form. Sample aliquots wereanalysed by a reverse-phase chromatography on a Supelcosil LC-18-DBcolumn (5 mm particle size, 30 cm×4.0 mm I.D., Supelco) with the mobilephase of ethanol:methanol 80:20 (v/v) at the flow rate of 1 ml/min.Absorbance at 275 and 290 nm was monitored.

A standard calibration curve of CoQ10 and Beckman System Gold Softwarewere utilized for data quantification. The amount of Coenzyme Q₁₀ wascalculated according to measured peak-areas of analysed samples. Thecalibration curve was prepared from a CoQ10 ethanol solution of aconcentration determined spectrophotometrically from its extinctioncoefficient of =14,200 at 275 nm. The ethanol solution of CoQ10 can bestored in sealed amber vials at -20° C.

The data presented in FIG. 2 show significantly improved (by 1.5 to 2fold) bioavailability of CoQ10 when given orally as a water solublecompositions in comparison with the oil formulation. Significantly, thekinetics of the uptake is much faster (plasma level peaked at about 3hr) and the maximal plasma levels achieved are also much higher (by 2fold) when CoQ10 is given in the water soluble form.

Example 16 illustrates the ability of cells to internalize thewater-soluble form of CoQ10.

Example 16 Intracellular Uptake of Coenzyme Q₁₀

The following experiments were designed to demonstrate the ability ofhuman keratinocytes to internalize exogenous CoQ10. Human skinkeratinocytes (ATCC CRL-8858), were cultured in SFM (serum free medium,Gibco BRL, cat no. 10724) supplemented with 0.2 ng/ml of EGF (epidermalgrowth factor), 30 mg/ml of bovine pituitary extract and differentconcentrations of CoQ10 which was added directly to the tissue culturemedium in a form of water soluble PCS formulation. The cells were grownfor up to 3 days under these conditions, then harvested and washedextensively. The cells (approximately 1-3×10⁶ /per sample) wereresuspended in ddH₂ O and were broken by an osmotic shock after afreezing and thawing cycle. CoQ10 was extracted by a propanolihexanesolvent mixture and its content was measured by the HPLC describedabove.

In order to obtain the mitochondrial fraction, the cells werehomogenized in a buffer containing 250 mM sucrose, 50 mM Tris-HCl pH7.4, 5 mM MgCl₂ and 1 mM EDTA. The homogenates were centrifuged at 800 gfor 10 min at 4° C. The supernatants were collected and were furthercentrifuged at 10,000 g for 10 min at 4° C. The pellets, representingcrude mitochondrial fractions, were resuspended in 100 ml of ddH₂ O,extracted with a propanol/hexane solvent mixture and processed for CoQ10analysis.

                  TABLE 10                                                        ______________________________________                                        Intracellular uptake of CoQ.sub.10 by cultured human keratinocytes            CoQ.sub.10 added                                                                           Total cellular CoQ.sub.10 content                                mg/ml of media                                                                             ng/10.sup.6 cells                                                ______________________________________                                        0 - control  10                                                               10           21.1                                                             100          49.6                                                             ______________________________________                                    

The data summarized in Table 10 show the intracellular uptake of CoQ10by human keratinocytes from the aqueous environment of tissue culturemedia. During the 3-day experimental period the intracellular content ofCoQ10 increased approximately 2-fold and 5-fold from the mediacontaining 10 mg/ml and 100 mg/ml of CoQ10 resuspended in the PCSformulation, respectively.

                  TABLE 11                                                        ______________________________________                                        Increased mitochondrial CoQ.sub.10 content in keratinocytes                   CoQ.sub.10 added           Total mitochondrial                                mg/ml of Total cellular CoQ.sub.10 content                                                               content                                            media    ng/10.sup.6 cells ng/10.sup.6 cells                                  ______________________________________                                         0       11                1                                                  10       69                9.8                                                ______________________________________                                    

The results shown in Table 11 demonstrate a significant increase ofCoQ10 content in the mitochondrial fraction of cells grown for 3 days inthe presence of 10 mg/ml of CoQ10. It is expected that, in addition toits function as lipid-soluble antioxidant, the elevated CoQ10 content,particularly in the mitochondria, will potentiate the efficiency of theenergy producing respiratory chain which is pivotal for the cellularviability.

Example 17 serves to demonstrate the ability of CoQ10 to increase theefficiency of mitochondrial respiratory chain to produce more ATP.

Example 17 Effects of Coenzyme Q₁₀ on Cellular ATP Content in Human NT2Cells

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were grown inDME medium (GiBco BRL) supplemented with 10% FBS (fetal bovive serum)and 10 mg/ml of CoQ10 which was added directly to the tissue culturemedium in a form of water soluble PTS formulation. The cells wereharvested at 24 hour intervals, resuspended in a buffer consisting of0.02M glycine, 0.05M MgSO₄, 0.004M EDTA, pH7.4 and aliquoted into 100 μLsamples.

The ATP content was measured using a luciferin-luciferasebioluminescence assay (Sigma, St. Louis, Mo.). Luciferase catalyses thedecarboxylation of luciferin and the hydrolysis of ATP to givepyrophosphate and oxyluciferin. These reactions result in emission oflight at 560 nm. The intensity of the emitted light is proportional tothe ATP concentration. The ATP assay was carried out by mixing 100 μL ofsample with 75 μL of a 0.5 mg/mL solution of luciferase-luciferin.Emitted light was detected using a Beckman LS 3801 scintillationcounter. A standard curve was prepared ranging from 10 to 100 pmols ofATP. ATP levels were expressed as pmols ATP/μg protein.

FIG. 3 shows the effect of PTS-CoQ10 composition on cellular ATP levelin NY2 cells. The ATP level was reproducibly higher in cells that werecultured for 3 days in the presence of water soluble PTS-CoQ10formulation. The data points represent mean values from three separateexperiments +/-SEM.

Example 18 shows protective effects of CoQ10 against hypoxia in humanneuroblastoma NT2 cells.

Example 18 Protection Against Hypoxia

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were seededinto 25 cm² tissue culture flasks (at a density of 0.3×10⁶ cells/ml) andwere grown for 3 days in DME medium (Gibco BRL) supplemented with 10%FBS and in the presence or absence of either 10 mg/ml of CoQ10-PTSformulation added directly to the tissue culture media. The flasks weresealed in an anaerobic chamber containing Gas Pak Plus gas generatorenvelopes and were incubated in the anaerobic conditions at 37° C. for17.5 hours in glucose-free medium. Following the hypoxic treatment thecells were placed again in the complete medium (with and without CoQ10or vitamin E) and were analysed either immediately (time 0) or werecultured for the additional 24 hr under the normoxic conditions. Cellviability was measured by trypan blue exclusion assay. The results areshown in FIG. 4.

The protective effects of CoQ10 against hypoxia in NT2 cells is clearlyevident. Approximately 50% less cells pre-treated for 3 days withPTs-CoQ10 died during the 24 hr recovery period from hypoxia as comparedto the cells which did not receive CoQ10.

Example 19 shows that CoQ10 is equally effective in protecting cellsagainst cell death triggered by hypoxia as vitamin E.

Example 19 Comparison of Coenzyme Q10 and Vitamin E Anti-oxidantFunction

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were seededinto 25 cm² tissue culture flasks (at a density of 0.3×10⁶ cells/ml) andwere grown for 3 days in DME medium (Gibco BRL) supplemented with 10%FBS and in the presence or absence of either 10 mg/ml of CoQ10-PTSformulation or the same concentration of vitamin E-PTS formulation,which were added directly to the tissue culture media. The flasks weresealed in an anaerobic chamber containing Gas Pak Plus gas generatorenvelopes and were incubated in the anaerobic conditions at 37° C. for17.5 hours in glucose-free medium. Following the hypoxic treatment thecells were placed again in the complete medium (with and without CoQ10or vitamin E) and were analysed either immediately (time 0) or werecultured for the additional 24 hr under the normoxic conditions. Cellviability was measured by trypan blue exclusion assay. The results aresummarized in FIG. 5.

In both experimental paradigms, the CoQ10 and Vitamin E treatments, thepercentage of death during the 24 hr recovery period from hypoxia isreduced by half. This demonstrates that the protective effects of CoQ10against hypoxia-induced cellular damage are comparable to those ofvitamin E, the antioxidant with proven efficacy.

Example 20 demonstrates the protective effects of CoQ10 againstischemia/reperfusion caused tissue damage. Although the animal modelused is the experimental paradigm of human stroke, the data is relevantfor any application where a tissue is damaged as a result ofischemia/reperfusion.

Example 20 Protective Effects of CoQ10 Against Ischemia/Reperfusion

Adult spontaneously hypertensive rats (SHR) weighting 225-250 g, whichreadily develop stroke characteristic lesions after short periods ofvessel occlusion, were used for the middle cerebral artery occlusionmodel (MCAO). The animals were anaesthetized and the right commoncarotid artery was permanently occluded. The skull was exposed and theright middle cerebral artery (MCA) was accessed through a 2 mm burr holein the skull. Under a dissecting microscope a microclip was applied tothe MCA vessel. After a 60 min occlusion period, the clip was removedand the wounds were closed. The body temperature was maintainedthroughout surgery and recovery at 37.5° C. using a rectal thermistorprobe connected to a heating lamp. CoQ10 (6 mg/kg) was administeredintravenously (neck vein) immediately after the removal of the clip. Theeffects of 60 min ischemia were quantitated by tetrazolium salt TTC(2,3,5-triphenyl,2H-tetrazolium chloride).

In order to quantitate the tissue damage, the brains were cut into 5 mmcoronal sections and were stained overnight with 2% TTC. The colouredformazan was extracted using acetonitrile and its concentration wasmeasured spectrophotometrically at 480 nm. The results as expressed asOD/g of dry brain tissue.

                                      TABLE 12                                    __________________________________________________________________________    Protective effects of CoQ.sub.10 against ischemia-induced brain               damage                                                                        Untreated animals                                                             Time                CoQ.sub.10 -treated animals                               after Left brain                                                                           Right brain                                                                          Time after                                                                          Left brain                                                                           Right brain                                  reperfusion                                                                         hemisphere                                                                           hemisphere                                                                           reperfusion                                                                         hemisphere                                                                           hemisphere                                   __________________________________________________________________________    sham  1250.0 +/-31.8                                                                       1239.0 +/-11.9                                                                       sham  1250.0 +/-31.8                                                                       1239.0 +/-11.9                               1 day 1208.0 +/-19.2                                                                       1018.0 +/-26.0                                                                       1 day 1206.0 +/-50.2                                                                       1200.0 +/-19.2                               2 days                                                                              1104.0 +/-36.2                                                                        898.3 +/-19.6                                                                       2 days                                                                              1084.9 +/-22.2                                                                       1060.9 +/-21.2                               __________________________________________________________________________

Tetrazolium salts are histochemical indicators of mitochondrialrespiratory enzymes and are used to detect tissue infarcts. TTC reactswith intact oxidative enzyme systems, such as succinate and NADHdehydrogenase, accepts electrons and becomes reduced to a coloredformazan which stains tissue red. By contrast, irreversible damagedmitochondria that do not have intact oxidative system cannot reduce TTCand the tissue remains white unstained. Accordingly, the healthier thetissue the higher the OD readings. Compare values obtained for brains ofsham operated control rats (approximately 1250 OD/g of tissue) and forbrains of experimental rats 898.3 OD/g tissue). The protective effectsof CoQ10 against ischemia-induced tissue damage are clearly evident,higher TTC content and, hence, the higher OD readings were obtained frombrains of animals which received CoQ10-treatment postischemically

Example 21 illustrate applicability and suitability of water solubleCoQ10 compositions in cosmetics.

Example 21 Water soluble CoQ10 Compositions in Cosmetics

                  TABLE 13                                                        ______________________________________                                        Preparation of cosmetics                                                      CoQ10-PCS-600           Final CoQ10                                           100 mg/ml               concentration                                         ______________________________________                                                   Classic Moisturizing Cream                                                    Oil of Olay                                                        1.5 ml      60 ml           0.25%                                             3.0 ml      60 ml           0.50%                                                        Classic Moisturizing Cream                                                    Oil of Olay                                                        1.5 ml      60 ml           0.25%                                             3.0 ml      60 ml           0.50%                                                        Dry Skin Moisturizer                                                          Pond's                                                             5 ml       200 ml           0.25%                                                        Moisturizing Body Lotion                                                      Vaseline                                                           7.5 ml     300 ml           0.25%                                                        Moisturizing Body Lotion                                                      Oil of Olay                                                        5 ml       200 ml           0.25%                                             ______________________________________                                    

Table 13 shows amounts of starting materials used to prepareCoQ10-containing skin products which were combined according to thefollowing procedure. The two components were mixed together at roomtemperature until they formed a uniform composition of a cream in whichCoenzyme Q10 remained water soluble. This was tested by adding water toa 0.2 ml sample of cream (total volume of 1 ml). The samples werevigorously vortexed and centrifuged to separate phases. The aqueousphase remained yellow since it contained dissolved CoQ10. The creamswere stable and no phase separation was observed even after severalmonths at room temperature.

These cosmetics were tested on 20 healthy volunteers over one yearperiod. All reported improved skin conditions, i.e. improved skinelasticity and reduction of wrinkles. None reported an adverse reaction.

Example 22 illustrates preparation of water soluble compositionscontaining polyene macrolide antibiotics.

Example 22 Preparation of Compositions of Anti-fungal Antibiotics

                  TABLE 14                                                        ______________________________________                                        Preparation of water-soluble formulations of anti-fungal                      antibiotics                                                                   ______________________________________                                        Candicidin            0.01          g                                         PCS-600 or PTS-600    0.02          g                                         THF/H.sub.2 O (8:2, v/v)                                                                            0.1           ml                                        H.sub.2 O             2.5           ml                                        Nystatin              0.01          g                                         PCS-600 or PTS-600    0.02          g                                         Glacial acetic acid   0.1           ml                                        H.sub.2 O             2.5           ml                                        Amphotericin B        0.01          g                                         PCS-600 or PTS-600    0.02          g                                         Methanol/Glacial acedic acid (2:1, v/v)                                                             0.3           ml                                        H.sub.2 O             2.5           ml                                        ______________________________________                                    

Table 14 shows amounts of starting materials used to preparewater-soluble compositions of antibiotics. Antibiotics and solubilizingagents were dissolved separately in an appropriate solvent or solventmixture. The solutions were combined and diluted with water. Solventsand excess of water were removed by evaporation under a reducedpressure. When glacial acetic acid was used as solvent, theco-evaporation with water was repeated several times. Compositionscontaining 20 mg/ml of antibiotic were obtained by solvent evaporation.They were stable and could be stored frozen for extensive periods oftime (8 months). These compositions could be also isolated, furtherconcentrated and sterilized by the technique described in Example 11.The final products were waxy, clear pellets which could be best storedfrozen, in vials sealed under argon (up to 8 months). They could bereconstituted in aqueous media (water or saline) to desiredconcentrations.

What is claimed is:
 1. A water-soluble composition comprising abioactive lipophilic compound and a solubilizing agent of the formula

    {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }.sub.p --Y

wherein: x is a residue of a hydrophobic moiety, selected from the groupconsisting of sterols, tocopherols, and derivatives thereof, y is aresidue of a hydrophilic moiety, selected from the group consisting ofpolyethers, polyalcohols, and derivatives thereof, p is 1 or 2, m is 0or 1, and n is an integer in the range 0≦n≦18,provided that: when p andm are equal to 1 and the hydrophobic moiety is cholesterol, n is greaterthan 4 and not equal to 8, and when p and m are equal to 1, and thehydrophobic moiety is (+)-α-tocopherol, n is not equal to
 2. 2. Acomposition according to claim 1, wherein the bioactive lipophiliccompound is selected from the group consisting of ubiquinones,ubiquinols, vitamins, provitamins, polyene macrolide antibiotics, andmixtures thereof.
 3. A composition according to claim 1, wherein thehydrophobic moiety is selected from the group consisting of cholesterol,7-dehydrocholesterol, campesterol, sitosterol, ergosterol, stigmasterol,and α-, β-, γ- , and δ-tocopherols.
 4. A composition according to claim3, wherein the the hydrophilic moiety is a polyether.
 5. A compositionaccording to claim 4, wherein the polyether is selected from the groupconsisting of polyethylene glycols.
 6. A composition according to claim5, wherein the polyethylene glycol has an average molecular weight offrom about 300 to about
 5000. 7. A composition according to claim 6,wherein the polyethylene glycol has an average molecular weight of fromabout 600 to about
 1000. 8. A composition according to claim 7, whereinthe hydrophobic moiety is selected from the group consisting ofcholesterol, β-sitosterol, α-(+)-tocopherol and α-(±)-tocopherol, and nis an integer from 2 to
 10. 9. A composition according to claim 8,wherein the bioactive lipophilic compound is selected from the groupconsisting of ubiquinones, ubiquinols, sterols, vitamin E, vitamin A,provitamin A, vitamin K, vitamin D, amphotericin-B, nystatin,candicidin, and mixtures thereof.
 10. A composition according to claim9, wherein the ubiquinione and ubiquinol are of the formula ##STR3##wherein R is ##STR4## and k is an integer of from 6 to
 12. 11. Acomposition according to claim 10, wherein k is equal to
 10. 12. Acomposition according to claim 9, further comprising a pharmaceuticallyacceptable, water-soluble additive.
 13. A composition according to claim12, wherein the additive is selected from the group consisting ofvitamin C, gelatin, a protein supplement, a polyethylene glycol having amolecular weight greater than about 5000, and mixtures thereof.
 14. Amethod of preparing a water-soluble composition according to claim 1,which method comprises the steps of:preparing a mixture of thelipophilic compound and the solubilizing agent in a predetermined molarratio, and heating the mixture at a temperature higher than therespective melting points of the lipophilic compound and thesolubilizing agent, for a time necessary to obtain a clear melt, andrecovering the water-soluble composition so obtained.
 15. A methodaccording to claim 14, wherein the molar ratio of the bioactivelipophilic compound and the solubilizing agent is from about 1:1 toabout 1:5.
 16. A method according to claim 15, wherein the molar ratiois about 1:2.
 17. A method of preparing a water-soluble compositionaccording to claim 1, which method comprises the steps of:dissolving thebioactive lipophilic compound and the solubilizing agent in apredetermined molar ratio in a water-miscible organic solvent, dilutingthe solution with a predetermined amount of water, and removing from thesolution the organic solvent and optionally an amount of water necessaryto achieve a desired concentration of the water-soluble composition. 18.A method according to claim 17, wherein the organic solvent and waterare removed by evaporation under reduced pressure.
 19. A methodaccording to claim 18, wherein the molar ratio of the bioactivelipophilic compound and the solubilizing agent is from about 1:1 toabout 1:5.
 20. A method according to claim 19, wherein the molar ratiois about 1:2.
 21. A method of purifying a water-soluble compositionaccording to claim 1, which method comprises the steps of:dissolving thewater-soluble composition in water, at a ratio of the composition towater not exceeding 1:2 by volume, heating the solution for a timenecessary to achieve separation of the water-soluble composition as aliquid phase, and separating the liquid phase from the hot solution,while keeping the temperature of the solution substantially unchanged.22. A method according to claim 21, wherein the separation of the liquidphase from the solution is achieved by centrifugation.
 23. Apharmaceutical or cosmetic formulation comprising a pharmaceutically orcosmetically effective amount of a bioactive lipophilic compound in theform of a water-soluble composition according to claim 1, saidformulation further comprising a pharmaceutically or cosmeticallyacceptable additive or vehicle selected from the group consisting ofsolvents, adjuvants, sweeteners, fillers, colourants, flavouring agents,lubricants, binders, moisturizing agents, preservatives and mixturesthereof.
 24. A formulation according to claim 23, wherein the bioactivelipophilic compound is selected from the group consisting ofubiquinones, ubiquinols, vitamins, provitamins, polyene macrolideantibiotics, and mixtures thereof.
 25. A formulation according to claim23, wherein the hydrophobic moiety is selected from the group consistingof cholesterol, 7-dehydrocholesterol, campesterol, sitosterol,ergosterol, stigmasterol, and α-, β, γ-, and δ-tocopherols.
 26. Aformulation according to claim 25, wherein the the hydrophilic moiety isa polyether.
 27. A formulation according to claim 26, wherein thepolyether is selected from the group consisting of polyethylene glycols.28. A formulation according to claim 27, wherein the polyethylene glycolhas an average molecular weight of from about 300 to about
 5000. 29. Aformulation according to claim 28, wherein the polyethylene glycol hasan average molecular weight of from about 600 to about
 1000. 30. Aformulation according to claim 29, wherein the hydrophobic moiety isselected from the group consisting of cholesterol, β-sitosterol,α-(+)-tocopherol, and α-(±)-tocopherol, and n is an integer from 2 to18.
 31. A formulation according to claim 30, wherein the bioactivelipophilic compound is selected from the group consisting ofubiquinones, ubiquinols, sterols, vitamin E, vitamin A, provitamin A,vitamin K, vitamin D, amphotericin-B, nystatin, candicidin, and mixturesthereof.
 32. A formulation according to claim 31, wherein theubiquinione and ubiquinol are of the formula ##STR5## wherein R is##STR6## and k is an integer of from 6 to
 12. 33. A formulationaccording to claim 32, wherein k is equal to
 10. 34. A formulationaccording to claim 23, in a form suitable for topical, oral, orparenteral administration.
 35. A formulation according to claim 34, inthe form of a capsule, tablet, caplet, granulate, suppository, sterilesolution, cream, lotion, gel, or ointment.
 36. A method of theprophylaxis or treatment of a medical disorder associated with oxidativetissue damages or mitochondrial dysfunctions, which method comprisesadministering to a human or a warm-blooded animal in need of suchprophylaxis or treatment a therapeutically effective amount of awater-soluble composition according to claim 1 comprising Coenzyme Q₁₀as the bioactive lipophilic compound, in conjunction with apharmaceutically acceptable diluent or carrier.
 37. A method accordingto claim 36, wherein the disorder is selected from the group consistingof heart diseases, ischemia/reperfusion tissue damages,neurodegenerative disorders and mitochondrial encephalomyolopaties. 38.A method of the prophylaxis or treatment of a medical disorder requiringCoenzyme Q₁₀ as an adjuvant, which method comprises administering to ahuman or a warm-blooded animal in need of such prophylaxis or treatmenta therapeutically effective amount of a water-soluble compositionaccording to claim 1 comprising Coenzyme Q₁₀ as a bioactive lipophiliccompound, in conjunction with a pharmaceutically acceptable diluent orcarrier.
 39. A method according to claim 38, wherein the disorder isselected from the group consisting of hypercholesteremia, infectiousdiseases and cancers.
 40. A method of the treatment of a fungalinfection, which method comprises administering to a human or awarm-blooded animal in need of such treatment a therapeuticallyeffective amount of a water-soluble composition according to claim 1comprising a macrolide polyene antibiotic as the bioactive lipophiliccompound, in conjunction with a pharmaceutically acceptable diluent orcarrier.
 41. A method of preparing a water-soluble composition ofCoenzyme Q₁₀, which method comprises the steps ofdirectly dissolving anon-crystallized product of synthesis of Coenzyme Q10 in a solubilizingagent of the formula

    {X--OOC--[(CH.sub.2).sub.n --COO].sub.m }.sub.p --Y

wherein: x is a residue of a hydrophobic moiety, selected from the groupconsisting of sterols, tocopherols, and derivatives thereof, y is aresidue of a hydrophilic moiety, selected from the group consisting ofpolyethers, polyalcohols, and derivatives thereof, p is 1 or 2, m is 0or 1, and n is an integer in the range 0≦n≦18,provided that: when p andm are equal to 1 and the hydrophobic moiety is cholesterol, n is greaterthan 4 and not equal to 8, and when p and m are equal to 1, and thehydrophobic moiety is (+)-α-tocopherol, n is not equal to 2, andrecovering the water-soluble composition so obtained.
 42. A methodaccording to claim 41, further comprising a purification of thewater-soluble composition, said purification comprising the stepsof:dissolving the water-soluble composition in water, at a ratio of thecomposition to water not exceeding 1:2 by volume, heating the solutionfor a time necessary to achieve separation of the water-solublecomposition as a liquid phase, and separating the liquid phase from thehot solution, while keeping the temperature of the solutionsubstantially unchanged.
 43. A method according to claim 42, wherein theseparation of the liquid phase from the solution is achieved bycentrifugation.
 44. A composition according to claim 4, wherein themolar ratio of the bioactive lipophilic compound and the solubilizingagent is from about 1:1 to about 1:5.
 45. A composition according toclaim 7, wherein the molar ratio of the bioactive lipophilic compoundand the solubilizing agent is about 1:2.